Download PDF

J. Embryol. exp. Morph. 81, 169-183 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
169
Development of the iris in the chicken embryo
II. Differentiation of the irideal muscles in vitro
By PATRICIA A. FERRARI AND WILLIAM E. KOCH
Department of Anatomy, School of Medicine, University of North Carolina,
Chapel Hill, NC 27514, U.S.A.
SUMMARY
The developmental capabilities of the iris rudiment in the chicken embryo, as well as the
role of tissue interactions in the differentiation of the iris, were investigated in vitro.
Sectors of the intact iris from 1\- through 9-day embryos (stages 32 through 35) lost their
morphological organization in vitro, but were capable of normal histodifferentiation. The
pigmentation of the epithelium increased, and muscle differentiation occurred. Developing
muscle was identified using immunocytochemistry with antiserum against chicken muscle
myosin; this procedure permitted positive identification of myoblasts, myotubes, and muscle
fibres in cultures in which histological features alone were equivocal. The proportion of irideal
explants which developed muscle increased with the age of the embryo, and correlated with
the incidence of epithelial buds and epithelial cells in the stroma.
Irideal mesenchyme from stage-32 through stage-35 embryos was already populated with
stromal epithelial cells when isolated, but growth and muscle differentiation in these cultures
compared poorly with that in the intact iris in vitro. Isolated irideal epithelium (stages 32
through 37) demonstrated even more limited muscle differentiation in vitro, suggesting
reciprocal interaction between irideal epithelium and mesenchyme during development.
Irideal epithelium was also cultured in direct association with non-irideal mesenchyme from
various embryonic organ rudiments, but muscle differentiation was not enhanced.
INTRODUCTION
Though the iris rudiment first becomes morphologically distinct with its outgrowth from the rim of the optic cup, its histodifferentiation is marked by the
development of the sphincter and dilator pupillae muscles and by the pigmentation of the irideal epithelium. Descriptive studies of the differentiation of the
sphincter and dilator muscles from the irideal epithelium (Brini, Porte &
Stoeckel, 1964; Imaizumi & Kuwabara, 1971; Lai, 1972a, b\ Tamura & Smelser,
1973; Ferrari & Koch, 1984), and of melanogenesis in the epithelial layers (ElHifnawi & Hinrichsen, 1975) can offer only limited insight into the factors which
operate during irideal differentiation. Nevertheless, in an ultrastructural study,
El-Hifnawi (1977) observed mesenchymal cells contacting the posterior irideal
epithelium during melanogenesis and concluded that differentiation of the
epithelium relied upon interaction with mesenchyme. While those observations
alone do not establish that epithelial melanogenesis depends upon mesenchymal
influence, experimental studies of many organ systems have shown that
170
P. A. FERRARI AND W. E. KOCH
epithelial-mesenchymal interactions are important for tissue differentiation (see
e.g. Koch, 1967; Lawson, 1974; Tyler, 1983). In addition, it has long been
recognized that differentiation of some epithelia is supported by heterologous
mesenchyme as well as homologous mesenchyme (Auerbach & Grobstein, 1958;
Grobstein, 1968).
Previous experimental studies of iris development have addressed primarily
the determination of the iris-ciliary body rudiment, which seems to require
presence of the lens (McKeehan, 1961; Stroeva, 1963, 1967; Genis-Galvez,
1966). In Stroeva's experiments, pigmentation in the inner layer of the optic cup
was taken as a definitive criterion of iris differentiation, though El-Hifnawi &
Hinrichsen (1975), have reported features of melanogenesis in the inner layer at
least 2 days prior to the outgrowth of the iris. These observations suggest that one
aspect of irideal histodifferentiation, the pigmentation of the posterior
epithelium, is initiated in the iris-ciliary body rudiment before the definitive iris
rudiment is formed. This is supported by the fact that pigmentation in the inner
epithelial layer is not a feature unique to the iris; this layer later becomes pigmented in a portion of the first ciliary process as well. For this reason, we chose to
emphasize the development of the sphincter and dilator muscles as representative of irideal histodifferentiation.
In this study, the developmental capabilities of the iris rudiment and isolated
epithelium and mesenchyme were examined in vitro. Identification of developing muscle was enhanced by using immunocytochemistry with myosin antiserum
(Ferrari & Koch, 1984). Our findings from study of the intact iris and isolated
irideal tissues suggest that epithelial-mesenchymal interactions function in the
differentiation of the iris.
MATERIALS AND METHODS
Fertilized chicken eggs from a commercial hatchery were incubated at 37-7 °C
in a humidified forced-draft incubator.
1. Dissection procedures
Chicken tissues
Eggs were cleaned with 80% ethanol; the embryos were transferred to
Tyrode's solution and staged according to their development (Hamburger &
Hamilton, 1951). Only tissues from embryos with the same degree of eye
development were used in a single experiment.
Irises were obtained from chicken embryos of 1\ through 9 days incubation
(stages 32 through 35); corneas were obtained from 8- and 9-day embryos (stages
33 and 35). The cornea was stripped from the eye using fine forceps, and transferred to a mixture of Tyrode's solution and horse serum (T/HS, 1:1 by volume).
Corneal mesenchyme was isolated by dissection and stored for later use in a
chamber of 5 % CO2 in air at room temperature. The remainder of the anterior
Differentiation of the iris in vitro
171
portion of the eye was removed intact and placed in T/HS. The anterior segment
of the eye was hemisected by a cut passing through the choroid fissure, and
before the halves were separated, a second cut was made bisecting each half. The
resulting quadrants were designated a through d starting with the quadrant
adjacent and temporal to the choroid fissure (a) and proceeding around the eye
to the quadrant adjacent and rostral to the choroid fissure {d; Fig. 1). At this
point, each quadrant included a portion of the iris, ciliary body, lens, and
vitreous body (Fig. 2). The sectors of the iris were then carefully cut from the
adjacent tissues and either reserved intact or placed into a cold (4°C) solution
of 3 % trypsin-pancreatin (3:1 by weight; Difco Laboratories, Detroit, MI) in
calcium- and magnesium-free Tyrode's solution (TP). The irideal epithelium and
mesenchyme were separated in this solution with gentle manipulation and rinsed
and stored in T/HS.
Mouse tissues
All mouse tissues were obtained from Brown Belt stock mice. Pregnancies
were timed from the day a copulation plug was detected; this was designated as
day 0 of gestation. On the appropriate day, a pregnant female was sacrificed by
cervical dislocation. The uterus was isolated and placed into sterile Tyrode's
solution. The embryos were then removed from the uterus and transferred to
T/HS. Dissections were carried out in T/HS; mesenchyme and epithelium of the
embryonic rudiments were separated in TP. Mesenchyme was isolated from 11day metanephric rudiments, from 13-day submandibular salivary gland rudiments, and from mandibular incisors of 16-day rudiments. Tissues were stored
in T/HS at room temperature in an atmosphere of 5 % CO2 in air.
2. Culture procedures
The culture procedure was similar to that devised by Grobstein (1956). The
culture assembly consisted of a disc of Millipore filter (THWP, 0-30 or 0-45 /im
porosity, 25 ± 5 jum thick; Millipore Corp., Bedford, MA) cemented to one side
of a plexiglass ring with two glass rods cemented to the opposite side of the ring.
The sterilized assemblies were placed over the wells of a depression slide within a
Petri dish. The tissue was placed upon a thin layer of congealed nutrient agar (1 %
agar in an equal volume of the culture medium) which coated the filter surface. In
some experiments, another thin layer of agar was added after the tissue was
positioned. The culture medium (Moscona, 1962) consisted of Eagle's basal
medium (Gibco, Grand Island, NY) supplemented with 1 % L-glutamine
(200 rriM, Gibco), 10 % foetal calf serum (Gibco), 3 % 11-day chicken embryo extract (Cameron, 1950) and 100 units/ml penicillin-streptomycin (Gibco). Each
well of the depression slide wasfilledwith medium so that thefluidlevel contacted
the filter. The tissues were incubated at 37-5 °C in a humidified atmosphere of 5 %
CO2 in air. The culture medium was changed or supplemented with fresh medium
every 48-72 h for the duration of the culture period, usually 7 or 8 days.
172
P. A. FERRARI AND W. E. KOCH
Culture of the intact iris
Sectors of the intact iris from 1\- through 9-day embryos (stages 32 through 35)
were cultured in vitro. At stage 32, only quandrants a and b were explanted,
whereas all quadrants from subsequent stages were used. The iris was positioned
with either its epithelial (posterior) or its mesenchymal (anterior) surface contacting the agar substratum.
Culture of isolated irideal tissues
Irideal mesenchyme was isolated from 1\- through 9-day embryos (stages 32
through 35) and cultured in vitro. Isolated irideal epithelium from 1\- through 11day embryos (stages 32 through 37) was also cultured.
Culture of irideal epithelium and non-irideal mesenchyme
Irideal epithelium from 1\- to 10-day chicken embryos (stages 32, 33, 35, and
36) was cultured in direct association with non-irideal mesenchyme from various
sources. Mesenchyme for the combined cultures was isolated from corneas of 8and 9-day chicken embryos (stages 33 and 35), and from the following organ
rudiments in mouse embryos: metanephric kidney of the 11-day embryo; submandibular salivary gland of the 13-day embryo and mandibular incisor of the
16-day embryo.
Cultures of the isolated irideal epithelium and cultures of the isolated nonirideal mesenchyme from the mouse embryos served as controls for the recombination experiments.
3. Histological procedures
All cultures were fixed in Bouin's fluid (Lillie, 1954), dehydrated in graded
ethanols, cleared in toluene, and embedded in Paraplast. Serial sections 5/zm
thick were cut and stained with haematoxylin and eosin or stained immunocytochemically. The primary antiserum used for the immunocytochemistry was rabbit antiserum to chicken skeletal muscle myosin (rA-cMyosin; Antibodies Inc.,
Davis, CA). The immunocytochemical staining procedure and the specificity
controls for the method and primary antiserum are described in an earlier report
(Ferrari & Koch, 1984). As in that study, we refer to positive immunocytochemical staining as muscle protein immunoreactivity (MPI).
RESULTS
Though outgrowth of the iris begins by stage 30 (Ferrari & Koch, 1984),
sectors of the iris were not large enough to adequately dissect from adjacent
tissues prior to stage 32. At stages 32 and 33, only quadrants a and b could be
isolated and maintained in culture. Beyond stage 33, all quadrants could be
Differentiation of the iris in vitro
173
isolated, but most cultures were set up with tissue from quadrants a or b to allow
for more valid comparison with earlier stages. We did not culture iris or irideal
mesenchyme from developmental stages greater than 9 days, or irideal
epithelium from stages beyond 11 days, in order to ensure that there was no
differentiation of muscle prior to explantation. In an earlier study on the
development of the iris in vivo (Ferrari & Koch, 1984), muscle protein immunoreactivity in the developing sphincter muscle was evident by 11 days of incubation and in the developing dilator muscle by 13 days.
Differentiation of the intact iris in vitro
Sectors of the dissected iris at stages 32 through 35 were fixed and sectioned
to serve as controls (Figs 3,4). Beginning at stage 32 in vivo, foci of the anterior
epithelium near the pupillary margin grow into the stroma forming epithelial
buds (Ferrari & Koch, 1984). Just prior to bud formation, cells of the anterior
epithelium at the site appear quite large and lie in disarray (Fig. 3). During
subsequent stages, more epithelial buds form around the iris, and as they enlarge, they extend farther into the irideal stroma (Fig. 4). Melanogenesis continues in the anterior epithelium during this period (stages 32 through 35), but
the posterior and marginal epithelium are still unpigmented at stage 35 (Fig. 4).
Table 1. Differentiation of muscle in irideal tissues in vitro
Stage
f
Tissue
•\
34
32
33
1;7*day) (74-8 day) (8 day)
A. Intact iris
Total number of cultures
Cultures with myotubes/
MPI
% Positive cultures
B. Isolated irideal
mesenchyme
Total number of cultures
No. of cultures with
myotubes/MPI
% of cultures with
myotubes/MPI
C. Isolated irideal epithelium
Total number of cultures
No. of cultures with
myotubes/MPI
% of cultures with
myotubes/MPI
9
9
3
7
33%
78%
6*
21
35
(9 day)
36
37
(10 day) (11 day)
36
17
35
81%
97%
7
14
34
It
3t
8
22
17%
43%
57%
65%
6
15
15
16
10
13
0
3
3
1
0
0
20%
20%
6%
0%
0%
0%
* Six cultures were explanted,but three disintegrated and did not survive the culture period.
t Single cells demonstrating MPI.
174
P. A. FERRARI AND W. E. KOCH
The morphology of the iris did not persist in vitro. The epithelium in cultures
from all stages usually lost its layered organization, though the pigmentation
increased substantially (Figs 5 and 6). Cultures from each stage developed
muscle with increasing frequency in explants from older embryos (Table 1, A).
Striated fibres were identified in cultures stained routinely (Fig. 5) or using
immunocytochemistry (Fig. 6). Myotubes were most evident following
Differentiation of the iris in vitro
175
immunocytochemical staining, but occasionally myotubes were seen which were
not positive for MPI. These myotubes were usually small and seen in cultures from
earlier stages. In general, MPI was present in the periphery of small myotubes
and throughout the cytoplasm in larger ones. Very large myotubes with abundant cytoplasm were common in explants from stage 35, and these could be
identified easily following routine staining (Fig. 7). Large myotubes were less
abundant in stage-34 explants and rare from stage-32 or stage-33 explants.
Differentiation of irideal mesenchyme in vitro
Sectors of isolated irideal mesenchyme were fixed and sectioned to serve as
controls (Figs 8, 9). At early stages, it was difficult to identify stromal epithelial
cells (Fig. 8), but at later stages, epithelial cells were present throughout the
stroma (Fig. 9). The development of irideal mesenchyme in vitro also varied with
the age of the explant. Mesenchyme from stage-32 and -33 (7J- to 8-day) embryos
failed to develop; the explants progressively diminished in size and often completely disintegrated. The explants from stages 32 and 33 did not develop
myotubes or muscle, although four cultures contained single cells demonstrating
MPI (Table 1, B, and footnote b). Explants of isolated mesenchyme from stage34 and -35 embryos usually survived the culture period intact, and in most cases
there was differentiation of muscle. Nevertheless, significantly fewer cultures of
isolated mesenchyme developed muscle compared to explants of the intact iris
(Table 1, A and B). Myotubes were identified by morphological characteristics in
conventionally stained sections (Fig. 10) or by immunocytochemistry (Fig. 11).
Note: Tissue preparation for the scanning electron micrographs (Figs 1 & 2) is
outlined in Ferrari & Koch, 1984.
Fig. 1. Scanning electron micrograph showing the right eye of a 10-day (stage-36)
chick embryo following removal of the cornea. The approximate position of the
choroid fissure is indicated (arrow); dotted lines delineate the quadrants formed after
further dissection. These are labelled a through d, starting with the quadrant (a)
adjacent and temporal to the choroid fissure and proceeding around the eye to the
quadrant (d) adjacent and rostral to the choroid fissure. x22.
Fig. 2. Scanning electron micrograph of a quadrant of the anterior segment of the
eye of a 9-day (stage-35) embryo. The cornea and lens have been removed; the iris
is indicated by the arrows. x62.
Fig. 3. Histological section of the iris from a 7£-day (stage-33) embryo. The anterior
epithelium near the pupillary margin has lost its epithelial organization (arrow).
X460.
Fig. 4. Histological section of the iris from a 9-day (stage-35) embryo clearly showing an epithelial bud (arrow). x460.
Fig. 5. Histological section of an iris from a 7j-day (stage-33) embryo after one week
in vitro. A striated muscle fibre is evident in the centre of the field. X1140.
Fig. 6. Immunoperoxidase-stained section of a culture of the iris from a 7|-day
(stage-33) embryo. The stained fibre (arrow) is multinucleated and lies in close
proximity to the pigmented epithelium. Antiserum: rA-cMyosin; dilution: 1:5000.
X460.
176
P. A. FERRARI AND W. E. KOCH
• v ;*r
'-,. ^
Fig. 7. Photomicrograph showing a culture of the iris from a 9-day (stage-35) embryo. Many large myotubes are seen in cross section. X460.
Fig. 8. Histological section of isolated irideal mesenchyme from a 7^-day (stage-32)
chick embryo prior to culture. x600.
Fig. 9. Isolated irideal mesenchyme from a 9-day (stage-35) chick embryo before
culture. An epithelial bud is indicated by the arrow; the centrally located stromal
epithelial cells have darkly stained nuclei. x600.
Fig. 10. Histological section of a culture of isolated mesenchyme from an 8-day
(stage-34) embryo. Myotubes are shown at the arrow. X540.
Fig. 11. Immunoperoxidase-stained preparation of cultured irideal mesenchyme
from a 9-day (stage-35) embryo. A stained fibre is obvious in the upper portion of
the field. The round granules at the extreme left and right sides of the field are
pigment granules. Antiserum: rA-cMyosin; dilution: 1:10000. X1140.
Fig. 12. Histological section of isolated irideal epithelium from a 7£-day (stage-32)
embryo prior to culture. The tissue rests upon a Millipore filter. X466.
Differentiation of the iris in vitro
177
Most cultures also contained small areas of sparsely pigmented cells (Fig. 11),
but this was not unexpected since some of the epithelial cells populating the
irideal stroma at the time of explantation included pigment granules (Fig. 9).
Differentiation of irideal epithelium in vitro
The complete separation of irideal epithelium from the stroma was assessed
during dissection, and also documented histologically in control sectors of
isolated irideal epithelium (Fig. 12). Cultures of isolated irideal epithelium from
Z'-jm&s&az^eZ* -r. 16
Fig. 13. Histological section of a culture of irideal epithelium from a 7^-day embryo.
A myotube is shown at the arrow. x620.
Fig. 14. Section of a culture of irideal epithelium with corneal mesenchyme; both
tissues are from a 9-day (stage-35) chick embryo. Note the abundant extracellular
material produced by the corneal mesenchyme. x370.
Fig. 15. Histological section through a culture of irideal epithelium from a 9-day
(stage-35) chick embryo with renal mesenchyme from an 11-day mouse. Nephrogenic
tubules (arrows) were formed in the mesenchyme. x430.
Fig. 16. Photomicrograph of a culture of irideal epithelium from a 9-day (stage-35)
chick embryo with mesenchyme from the salivary gland of a 16-day mouse.
Myotubes (arrow) are visible in the mesenchyme. x430.
178
P. A. FERRARI AND W. E. KOCH
1\- through 11-day embryos (stages 32 to 37) usually remained healthy and
showed significant increases in size and pigmentation. The epithelium from
certain stages was capable of limited muscle differentiation (Table 1, C).
Myotubes were demonstrated in explants from stages 33 and 34 and in a single
culture from stage-35 tissue. Occasionally large myotubes with multiple nuclei
were evident (Fig. 13), but most myotubes were small, and could be identified
only by MPI. Myotubes or muscle were never seen in explants from stages 32,
36, or 37.
Differentiation of irideal epithelium and non-irideal mesenchyme in vitro
Isolated irideal epithelium from 7f- to 10-day embryos (stages 32, 33, 35 and
36) was cultured in direct association with non-irideal mesenchyme from chick
cornea and murine incisor, kidney and salivary gland. The non-irideal mesenchyme was also cultured in isolation to serve as controls for the combined explants. All cultures were assessed for the presence of muscle.
In the recombinant cultures, both the irideal epithelium and the nonirideal mesenchyme from all sources remained healthy, and increased in size
during the culture period. As in the cultures of irideal epithelium alone, the
irideal epithelium in the recombinant cultures became darkly pigmented and
usually did not maintain an epithelial organization apparent in sections (Figs
14-16).
The combined explants of irideal epithelium with mesenchyme from chick
cornea or murine incisor or kidney never developed myotubes or muscle, and in
general, the effect of the recombination was more apparent on the development
of the mesenchyme. In the 16 explants of irideal epithelium combined with
corneal mesenchyme, the mesenchyme produced abundant extracellular
material (Fig. 14). The amount of extracellular material in control cultures of
isolated corneal mesenchyme appeared unchanged during the culture period.
Incisor mesenchyme was explanted with irideal epithelium in 12 cultures, and the
mesenchyme remained healthy and increased in size but without differentiation.
In contrast, isolated incisor mesenchyme in control cultures was poorly maintained in vitro and diminished in size. The control cultures of isolated renal
mesenchyme also failed to develop in vitro, but when renal mesenchyme was
combined with irideal epithelium, kidney tubules formed in the mesenchyme in
all 12 cultures (Fig. 15).
Irideal epithelium was also cultured with mesenchyme from the murine salivary gland rudiment. In 15 of 26 explants, the mesenchyme contained myotubes
which could be identified histologically (Fig. 16) or by immunocytochemistry.
However, myotubes also developed in four of seven control cultures of isolated
salivary mesenchyme. In many cases, living cultures of salivary gland mesenchyme alone or combined with irideal epithelium could be observed to contract.
Contractile activity was never observed in any irideal cultures which did not
contain salivary mesenchyme.
Differentiation of the iris in vitro
179
DISCUSSION
The results of these studies indicate that the intact iris rudiment will grow and
differentiate in vitro, but without retaining the same level of organization that is
present in vivo. Nevertheless, the pigmentation of the epithelium increases and,
with increasing frequency in older explants, muscle differentiation occurs. These
experiments do not establish when determination of the iris occurs, i.e. when the
tissues first exhibit an inherent ability to differentiate. Though the pigmentation
of the inner layer of the optic cup is a criterion which has been used in some
studies to indicate the determination of the iris (Stroeva, 1963,1967) it has never
been shown that the formation of the iris rudiment, and its subsequent differentiation, necessarily follows that step. Melanogenesis occurs in the inner
epithelial layer of the iris-ciliary body rudiment prior to outgrowth of the iris (ElHifnawi & Hinrichsen, 1975) and, in the adult chicken, the inner epithelium of
both the iris and a portion of the first ciliary process are pigmented (Ferrari,
personal observation). Thus pigmentation of this layer is not a unique feature of
irideal determination or histodifferentiation, unlike the differentiation of the
sphincter and dilator muscles from the irideal epithelium.
In this study, 7|-day embryos were the youngest from which the intact iris
rudiment was isolated, and one third of these explants formed muscle in vitro.
The proportion of cultures which formed muscle progressively increased with
explants from older embryos. This pattern corresponds to the increase in size and
distribution of epithelial buds over the same period, and it seems likely that the
ability of the irideal explant to form muscle in vitro is related to the incidence of
buds and stromal epithelial cells.
Explants of isolated irideal mesenchyme also showed features of differentiating muscle at each age explanted, though a lower proportion of positive cultures
was seen when compared with explants of the intact iris. Furthermore, there
were fewer myotubes and immunoreactive cells in the cultures of isolated mesenchyme than in the cultures of the intact iris. It is possible that during culture of
the intact rudiment the epithelium continually contributes cells which differentiate into muscle, and the more extensive differentiation of muscle in the intact
rudiment in vitro may be due to the fact that there were more muscle cell precursors. Other differences between the cultures of the isolated mesenchyme and the
intact rudiment indicate that the mesenchyme requires the presence of the
epithelium for its maintenance as a tissue. At early stages, irideal mesenchyme
frequently was not maintained in vitro', the tissue simply disintegrated during the
culture period. Since quadrants of the iris vary greatly in size between 1\- and
9-day embryos, the mass of the younger explants was increased by pooling
mesenchyme, but without improvement in survival or differentiation. It is likely
that more cultures from older embryos remained intact not because of the larger
size of the original explant, but because of the presence of more stromal
epithelial cells. The epithelium of the iris and the stromal epithelial cells seem
180
P. A. FERRARI AND W. E. KOCH
to support the maintenance of the mesenchyme as a tissue, while at the same
time, the mesenchyme enhances the differentiation of muscle.
The latter point was considered by examining the ability of the irideal
epithelium to give rise to muscle in the absence of the irideal mesenchyme. Since
it is known from the study of various embryonic organs that epithelia are dependent upon an association with mesenchyme for continued development (Grobstein, 1968), it was unexpected that isolated epithelial cultures would form
muscle in vitro. However, very few of the cultures developed muscle, and then
only from embryos between stages 33 and 35. It remains unclear why the
epithelium at only certain stages expressed the capability for muscle differentiation in the absence of mesenchyme. One possibility is that the epithelium itself
changes after stage 35, and develops an absolute requirement for mesenchyme.
It is also possible that the dissection and separation of the tissues changes with
the growth of the iris and the enlargement of the epithelial buds, allowing more
complete separation of the ridge of the sphincter muscle precursor cells from the
epithelial layer at later stages. Dissection alone would not account for the complete failure to develop muscle beyond stage 35, since the dilator muscle differentiates from the entire anterior epithelium. In either case, it is clear that the
irideal mesenchyme has an important effect upon the differentiation of muscle
in the iris. Though the epithelial cultures remained healthy in the absence of
mesenchyme, the proportion which developed muscle was very low compared to
cultures of the intact iris or of mesenchyme alone.
The most direct test for interactions of the epithelium and mesenchyme in the
development of an organ would be to recombine both tissues in vitro. However,
since the irideal mesenchyme already possesses a population of epithelial cells,
the differentiation of these stromal epithelial cells would mask any response of
the epithelium to the mesenchyme. Consequently, muscle differentiation was
assessed by culturing irideal epithelium with non-irideal mesenchyme from chick
and mouse embryos. Numerous studies have established that morphogenesis and
histogenesis can be supported in vitro by recombining epithelium and mesenchyme from different organs within the same species (e.g., chick-chick recombinations, see Coulombre & Coulombre, 1971; Tyler, 1983) or from different
species (e.g., chick-mouse recombinations, see Houissant & Le Douarin, 1968;
Coulombre & Coulombre, 1971; Kollar & Fisher, 1980; chick-rabbit recombinations, see Propper, 1969). In testing the tissue requirements for differentiation
in irideal epithelium, non-irideal mesenchyme was chosen from both chick and
mouse embryos.
Chick corneal, and murine incisor and metanephric mesenchyme were chosen
for the recombination experiments because each supports the differentiation of
an epithelium that does not form muscle in vivo. Corneal and incisor mesenchyme are derivatives of neural crest and thus similar to irideal mesenchyme
which is also derived from neural crest (Johnston et al., 1979). Corneal mesenchyme was also chosen for its ability to form abundant collagenous extracellular
Differentiation of the iris in vitro
181
material, since it has been suggested that components of extracellular matrices
influence muscle differentiation in vitro (Konigsberg, 1970; Ketley, Orkin &
Martin, 1976; Yamada, Olden & Hahn, 1980). In each of these cases, the nonirideal mesenchyme failed to induce muscle differentiation in the irideal
epithelium. Nevertheless, other interactions between the recombined tissues
were observed. Irideal epithelium enhanced the growth of incisor mesenchyme
and supported tubule formation in kidney mesenchyme. Grobstein & Parker
(1958) have reported that metanephrogenic mesenchyme will form tubules
when grafted into the anterior chamber of an adult eye, but neither iris nor
cornea from the adult eye has been shown to support tubule formation in vitro
(Grobstein, 1955). However, kidney mesenchyme does form tubules in vitro
in response to embryonic dorsal spinal cord (Auerbach & Grobstein, 1958) and
like the spinal cord, the irideal epithelium is derived from a neuroepithelium.
Murine salivary gland mesenchyme was selected for recombination because of
its association with and possible influence upon an epithelium which forms
myoepithelial cells in vivo. It has not previously been reported that isolated
salivary mesenchyme forms muscle in vitro, but in this study, myotubes were
observed in most explants of salivary mesenchyme whether cultured alone or
with irideal epithelium. Other studies of isolated salivary mesenchyme from
13-day mouse embryos (Grobstein, 1953) or from 14-day mouse embryos
(Lawson, 1974) have not reported the formation of myotubes. In the present
study, muscle differentiation may have been enhanced by the agar substratum
which prevented spreading of the explant. Lawson (1974) used a similar
substratum, but he cultured salivary mesenchyme from the 14-day mouse
embryo. It may be that the capability for myotube formation in isolated salivary
gland mesenchyme does not extend beyond the 13-day embryo, or simply that
in Lawson's study too few cultures were examined to detect a limited capacity
for muscle differentiation. In any case, murine salivary gland mesenchyme was
apparently unaffected by culture with irideal epithelium, whereas the development of chick corneal mesenchyme and murine incisor mesenchyme was
enhanced. In the case of murine metanephric mesenchyme, the irideal
epithelium clearly permitted the differentiation of kidney tubules. Despite
these interactions, the non-irideal mesenchyme that we tested did not support
differentiation of irideal muscle.
In conclusion, these experiments establish that even in the absence of
normal morphogenesis, the intact iris is capable of histodifferentiation in vitro.
Neither isolated irideal mesenchyme nor irideal epithelium demonstrated the
capability for muscle differentiation exhibited by the intact iris, and this
suggests that epithelial-mesenchymal interactions function in irideal differentiation in vitro.
The authors are grateful to Ms Daynise Skeen for technical assistance. This investigation
was supported in part by USPH Research Grant DE 04580 from the National Institute of
Dental Research.
182
P. A. FERRARI AND W. E. KOCH
REFERENCES
R. & GROBSTEIN, C. (1958). Inductive interaction of embryonic tissues after
dissociation and reaggregation. Expl Cell Res. 15, 384-397.
BRINI, A., PORTE, A. & STOECKEL, M. E. (1964). D6veloppement ultrastructural des muscles
iriens chez l'embryon de poulet. Bull. Mem. Soc. Fr. Ophthalmol. 77, 488-497.
CAMERON, G. (1950). Tissue Culture Technique. 2ndEdition, p. 55. New York: Academic Press.
COULOMBRE, J. L. & COULOMBRE, A. J. (1971). Metaplastic induction of scales and feathers
in the corneal anterior epithelium of the chick embryo. Devi Biol. 25, 464-478.
EL-HIFNAWI, E. (1977). Interaction between mesenchymal cells and the posterior iris
epithelium in chick embryos. Anat. Embryol. 151, 109-118.
EL-HIFNAWI, E. &HINRICHSEN,K. (1975). Melanogenesis in the pigment epithelium of chicken
embryos. I. Topogenesis of pigment in the iris anlage. Anat. Embryol. 147, 177-187.
FERRARI, P. A. & KOCH, W. E. (1984). Development of the iris in the chicken embryo. I. A
study of growth and histodifferentiation utilizing immunocytochemistry for muscle differentiation. /. Embryol. exp. Morph. 81, 153-167.
GENIS-GALVEZ, J. M. (1966). Role of the lens in the morphogenesis of the iris and cornea.
Nature 210, 209-210.
GROBSTEIN, C. (1953). Epithelio-mesenchymal specificity in the morphogenesis of mouse submandibular rudiments in vitro. J. exp. Zool. X2A, 383-413.
GROBSTEIN, C. (1955). Tissue interaction in the morphogenesis of mouse embryonic rudiments
in vitro. In: Aspects of Synthesis and Order in Growth, (ed. D. Rudnick), pp. 233-256.
Princeton: Princeton University Press.
GROBSTEIN, C. (1956). Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Expl Cell Res. 10, 424-440.
GROBSTEIN, C. (1968). Developmental significance of interface materials in epitheliomesenchymal interaction. In: Epithelial-MesenchymalInteractions, (ed. R. Fleischmajer & R. E.
Billingham,), pp. 173-176. Baltimore: Williams and Wilkins.
GROBSTEIN, C. & PARKER, G. (1958). Epithelial tubule formation by mouse metanephrogenic
mesenchyme transplanted in vivo. J. natn. Cancer Inst. 20, 107-119.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HOUISSANT, E. & LE DOUARIN, N. (1968). La differentiation de l'endoderme hepatique de
poulet associe au mesenchyme metanephritique de l'embryon de souris. C.r. hebd. Sdanc
Acad. ScL, Paris 267, 201-202.
IMAIZUMI, M. & KUWABARA, T. (1971). Development of the rat iris. Invest. Ophthalmol. 10,
733-744.
AUERBACH,
JOHNSTON, M. C , NODEN, D. M., HAZELTON, R. D., COULOMBRE, J. L. & COULOMBRE, A. J.
(1979). Origins of avian ocular and periocular tissues. Expl Eye Res. 29, 27-43.
N., ORKIN, R. W. & MARTIN, G. (1976). Characterization of chick embryo skeletal
muscle collagen. Expl Cell Res. 99, 261-268.
KOCH, W. E. (1967). In vitro differentiation of tooth rudiments of embryonic mice. I. Transfilter interaction of embryonic incisor tissues. /. exp. Zool. 165,155-170.
KOLLAR, E. J. & FISHER, C. (1980). Tooth induction in chick epithelium: Expression of
quiescent genes for enamel synthesis. Science 207, 993-995.
KONIGSBERG, I. R. (1970). The relationship of collagen to the clonal development of embryonic
skeletal muscle. In: Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 3,
(ed. E. A. Balazs), pp. 1779-1810. New York: Academic Press.
LAI, Y.-L. (1972a). The development of the sphincter muscle in the iris of the albino rat. Expl
Eye Res. 14, 196-202.
LAI, Y.-L. (1912b). The development of the dilator muscle in the iris of the albino rat. Expl
Eye Res. 14, 203-207.
LAWSON, K. A. (1974). Mesenchyme specificity in rodent salivary gland development: The
response of salivary epithelium to lung mesenchyme in vitro. J. Embryol. exp. Morph. 32,
469-493.
KETLEY, J.
Differentiation of the iris in vitro
183
R. D. (1954). Histopathologic Technic and Practical Histochemistry, p. 46. New York:
Blakiston.
MCKEEHAN, M. S. (1961). The capacity for lens regeneration in the chick embryo. Anat. Rec.
141, 227-230.
MOSCONA, A. A. (1962). Analysis of cell recombination in experimental synthesis of tissues
in vitro. J. cell. comp. Physiol. 60, 65-80.
PROPPER, A. (1969). Competence de l'epiderme embryonnaire d'Oiseau vis-a-vis de l'inducteur mammaire mesenchymateux. C.r. hebd. Sianc Acad. Sci., Paris. 268, 1423-1426.
STROEVER, O. G. (1963). The role of the lens epithelium in the induction of iris and ciliary body
tissue. Dokl. Acad. Nauk. SSSR 151, 977-980.
STROEVA, O. G. (1967). The correlation of the processes of proliferation and determination
in the morphogenesis of iris and ciliary body in rats. /. Embryol. exp. Morph. 18,269-287.
TAMURA, T. & SMELSER, G. K. (1973). Development of the sphincter and dilator muscles of
the iris. Arch. Ophthalmol. 89, 332-339.
TYLER, M. S. (1983). Development of the frontal bone and cranial meninges in the embryonic
chick: An experimental study of tissue interactions. Anat. Rec. 206, 61-70.
YAMADA, K. M., OLDEN, K. & HAHN, L.-H. E. (1980). Cell surface protein and cell interactions. In: The Cell Surface: Mediator of Developmental Processes, (ed. S. Subtelny & N. K.
Wessells), pp. 43-78. New York: Academic Press.
LILLIE,